Supplementary MaterialsSupplementary information 41598_2017_3799_MOESM1_ESM. MEKK13 directly to cultured endothelial cells does not influence endothelial cell migration or proliferation. The enhanced tube formation can be seen for up to 10?hours where purchase E 64d after the effect decreases. It is shown that the antibody-binding site is located on the coil 2 domain of vimentin. To our knowledge this is the first study that demonstrates an enhanced tube formation by binding vimentin in a 2D matrigel assay under purchase E 64d normoxic conditions. Introduction The intermediate filament protein vimentin exert important intracellular functions, regulating processes like cell migration and sustaining cell integrity. The importance of vimentin-mediated processes was underestimated for years, mainly because vimentin deficient (biotinylation protocol developed by the group of Dario Neri28, a selection of antibodies was performed against biotinylated proteins from HUVEC cells. In total 384 clones were picked and monoclonal phage antibodies were produced followed by sceening for their binding to HUVEC and HMEC-1 cells by phage antibody ELISA (Supplementary Fig.?S1). One of the antibodies selected for further investigation was LOB7. Initial characterisation by ICC showed that LOB7 bound more to old ASF-2 (passage 52) than to young ASF-2 (passage 10) (Supplementary Fig.?S1). LOB7 binds vimentin The scFv holding a His tag motif was immobilised on Ni-NTA magnetic resin beads and used to precipitate proteins directly from sonicated HUVEC lysates. Precipitated proteins were separated by SDS-PAGE, visualised by silver stain, and bands of interest were excised from the gel and analysed by mass spectrometry (Supplementary Fig.?S2). Vimentin was identified as the top hit from the mass spectrometry analysis (Supplementary Tabel S1). Some of the residual sample of precipitated proteins was analysed by western blot using the mouse monoclonal anti vimentin antibody V9 (Fig.?1a). This confirmed the presence of vimentin in the sample of proteins precipitated from HUVEC lysates by the use of LOB7. Accordingly, phage antibody ELISA was performed on serial diluted recombinant vimentin. A clear signal was observed (Fig.?1b). To further validate the binding of LOB7 to vimentin, a control ELISA was performed where laminin and skimmed milk powder was included as negative controls (Supplementary Fig.?S3). Open in a separate window Figure 1 Characterisation of LOB7. Top panel: (a) The commercial mouse anti-vimentin V9 was used to identify vimentin in the sample of immuno-precipitated proteins. (b) LOB7 presented on phage was tested against vimentin in ELISA. A dilution series of vimentin was coated in the wells of an ELISA plate and detected using the same amount of LOB7 presented on phage. (c) Western blot using the commercial mouse anti-vimentin V9 and LOB7 respectively on cytoplasmatic extracts from HUVEC cells and extracts from the extracellular matrix (Matrigel). Additionally, the specificity of the LOB7 antibody was assessed by western blot analysis of HUVEC lysates and growth factor reduced matrigel. The anti-vimentin V9 antibody was used as a positive control. As can be seen, the proteins detected by LOB7 have the same apparent size as those detected by V9 (Fig.?1c). To map the binding of the antibody, seven fragments covering different areas of vimentin were constructed by PCR and expressed in (Fig.?2). Long unstructured protein sequences, which are often seen in purchase E 64d eukaryotic cells, are very susceptible to degradation29. Hence, to protect some of the intrinsically unstructured domains of vimentin against degradation, all seven fragments were expressed in conjugation with the pagP beta-barrel membrane protein30. It was shown that LOB7 binds to the coil 2 domain of vimentin, while the mouse anti-vimentin V9 binds to the tail region of vimentin. Open in a separate window Figure 2 Testing specificity of LOB7 and V9 by western blotting. To test the specificity of LOB7 and the mouse monoclonal anti-vimentin antibody V9,.